WO2011011962A1

WO2011011962A1 - Method for preparing complex multi-layer tissue organ precursor

Info

Publication number: WO2011011962A1
Application number: PCT/CN2010/000439
Authority: WO
Inventors: 王小红; 张人佶; 颜永年; 隋少春
Original assignee: 清华大学
Priority date: 2009-07-31
Filing date: 2010-04-06
Publication date: 2011-02-03
Also published as: CN101623515A

Abstract

A method for preparing a complex multi-layer tissue organ precursor is composed of following steps. Synthetic polymer solution is filled into an inner mold to form an inner scaffold. Then cellular matrix solution is filled into the space between the inner scaffold and each middle mold to form cellular matrix middle layer, which is physically or chemically crosslinked to form a multi-layer structure between the inner scaffold and the cellular matrix middle layer. The multi-layer structure is put into an outer mold, and the synthetic polymer solution is filled into the interspace between the multi-layer structure and the outer mold to form an outer synthetic polymer material layer. At last, a complex multi-layer tissue organ precursor is prepared. The molds of each layer are designed according to the structural characteristics of a certain tissue organ to be prepared.

Description

Method for preparing complex tissue organ precursor with multi-layer structure

Technical field

The invention belongs to the technical field of artificial manufacturing of biological tissues and organs, and particularly relates to a method for preparing a tissue organ precursor by using a synthetic high molecular material and a cell matrix material, and belongs to the field of biological tissue engineering technology.

Background technique

There are more than 10 million patients suffering from tissue defects or organ failure every year in the world. In the United States alone, about 8 million patients are treated surgically each year. However, living donor organs are limited, and existing mechanical devices do not have all the functions of organs, and cannot prevent the patient's condition from further worsening. Accordingly, Tissue Engineering technology for the purpose of improving the treatment level of such diseases has emerged. Tissue engineering is a high-tech discipline that is produced by multiple disciplines such as biology, medicine, materials science, and engineering. Its meaning is to apply the principles and techniques of life sciences and engineering, based on the correct understanding of the relationship between the structure and function of the normal and pathological states of mammals, research and development for the repair, maintenance and promotion of various human bodies. Biological substitutes for function and morphology after tissue or organ injury [Merem RM. Med & Biol Eng & Comput. 1992; 30:8-12]. In the past ten years, scientists have used tissue engineering techniques to use small amounts of normal cells in the human body to reproduce in vitro, to obtain the organs that the patients need, have the same function, and there is no rejection reaction, which has yielded gratifying results, many recent The established biotechnology company is investing heavily in commercialization. In the United States, a $4 billion industry has been formed and is growing at a rate of 25% per year. For example, the Pro Osteon coral bone graft material has a production value of more than 10 million US dollars [Miller M, Biotech Bioeng, 1996; 50: 4347-4348] ₀ but the existing tissue engineering technology faces many difficulties and limitations, and the success of the tissue engineering application research institute is In those structures and physiological functions are relatively simple Organs such as bones, cartilage, and skin. Traditional tissue engineering methods generally prepare structural scaffolds. During the cell culture process, the upper cells consume most of the oxygen and nutrients, which restricts the diffusion of these components to the bottom layer, thereby limiting the migration of cells to the deep layers of the scaffold. The method of preparing the stent first and then culturing the cell is time consuming and laborious, and the cell may have been deformed and aged in the process of migrating into the stent, failing to meet the requirements for timely treatment of the clinical patient.

The invention patent of application No. 200410091552.4 is (1) centrifugally separating different kinds of seed cells or gene suspensions and uniformly mixing them with a solution containing 1 to 40% of a matrix material, and (2) biodegradable polymer materials. Or a solution of the complex with heparin, using a computer-assisted rapid prototyping technique, a coating method or a one-time infusion forming technique, freeze-drying to form a three-dimensional structural support of different parts of the tubular tissue and organ, and finally step (2) The prepared tubular structure is inserted into the pipe-like three-dimensional structure prepared in the step (1), and a layer of degradable polymer film is wrapped. The patented method produces an organ precursor that is not tightly bonded between different layers of material. The invention patent No. 200510063378.7 provides a complex organ precursor with a culture device and a method for constructing and cultivating the same, aiming at directly assembling the cell and extracellular matrix bionic material by computer modeling and cell assembly technology. In a three-dimensional structure, a complex organ precursor is subjected to a three-dimensional stress field by a culture device to establish a connection between cells and induce growth, thereby forming a functional tissue, thereby causing the transition of such organ precursors to a functional organ, thereby achieving repair and regeneration. the goal of. The culture product of this patented method cannot be directly linked to the vasculature of the body.

Conventional stent preparation techniques are difficult to form a nutrient supply channel with branches. At the same time, traditional tissue engineering techniques can not meet the needs of accurately locating and positioning different cells in space to construct functional gradient structures of complex tissue organs.

Summary of the invention

The object of the present invention is to provide a method for preparing a complex tissue organ precursor, aiming at the previous work, using a step-by-step mold/extraction method to achieve accurate positioning of cells and scaffold materials in space, utilizing The principle of mold combination and polymer solidification forming realizes the reconstruction of complex tissues and organs, overcomes the shortcomings of tissue engineering in the three-dimensional scaffold, which requires long time, uneven cell distribution, and difficult to penetrate into deep structures, thus achieving repair. The purpose of regeneration.

The technical solution of the present invention is as follows:

A method for preparing a complex structure organ precursor of a multilayer structure, the method is carried out as follows:

(1) dissolving the synthetic polymer material in an organic solvent to prepare a synthetic polymer solution having a mass percent concentration of 1% to 30%;

(2) mixing the seed cell suspension with a natural polymer solution having a mass concentration of 10% to 30% to prepare a cell matrix solution, wherein the density of the seed cells in the cell matrix solution is lx lO^l xlO ⁹ /mL;

(3) injecting the synthetic polymer solution tank into a pre-designed inner mold, and then extracting the organic solvent in the synthetic polymer solution with water or PBS solution, and removing the inner mold to form a single-layer synthetic polymer material inner support;

(4) According to the structure and characteristics of the tissue and organs to be prepared, intermediate molds of different diameters are designed, the intermediate mold is at least one, and the first intermediate mold is sleeved on the outside of the single-layer synthetic polymer material, and then the corresponding The cell matrix solution tank is injected between the inner stent and the first intermediate mold, and the natural polymer in the cell matrix solution is crosslinked by physical or chemical crosslinking, and the first intermediate mold is removed to form a stable synthetic polymer material. a two-layer structure of the intermediate layer of the stent and the first cell matrix; then the second intermediate mold is sleeved on the outside of the two-layer structure, and the corresponding cell matrix solution tank is injected between the second intermediate mold and the two-layer structure, via physical Or chemical crosslinking method, cross-linking the natural polymer in the cell matrix solution, removing the second intermediate mold to form a stable synthetic polymer material inner scaffold, the first cell matrix intermediate layer and the second cell matrix intermediate layer three-layer structure Increasing the intermediate layer of the cell matrix layer by layer according to the above method to form a stable multilayer structure; (5) loading the above-mentioned multilayer structure into a pre-designed outer mold, and then pouring the synthetic polymer solution into the gap between the multilayer structure and the outer mold to form an outer layer of synthetic polymer material;

(6) Extracting the organic solvent in the outer layer of the synthetic polymer material with water or PBS solution, and removing the outer mold, thereby preparing a complex structure organ precursor of a multilayer structure.

The synthetic polymer material is a composite of one or both of polyurethane, lactic acid and glycolic acid copolymer, polylactic acid and copolymers thereof and polyester.

The natural polymer is at least one of gelatin, fibrinogen, collagen, chitosan, sodium alginate, hyaluronic acid, and fibronectin.

The organic solvent used in the step (1) for dissolving the synthetic polymer material is tetraethylene glycol, ethylene glycol, isopropanol or 1, 4-dioxane.

The solvent of the natural polymer solution in the step (2) is water, physiological saline, PBS solution, 0.09 M sodium chloride, 3-hydroxymethylcarbamidine hydrochloride solution or cell culture solution having a pH of 6 to 8.

A cryopreservation agent having a volume percentage of 1% to 30% is also added to the cell substrate solution.

The cryoprotectant is a mixture of one or both of glycerin, dimethyl sulfoxide, ethylene glycol and dextran.

A percentage by volume of 0.001% to 0.1% of cell growth factor is also added to the cell substrate solution. The cell growth factor is an endothelial cell growth factor, a cell transfer factor or a hepatocyte growth factor.

One or two anticoagulant materials may be added to the synthetic polymer solution or the cell matrix solution, wherein the weight of the anticoagulant material and the volume percentage of the solution are 0.1 to 10%.

The anticoagulant material is heparin, sodium citrate or paclitaxel. The selection of seed cells in the intermediate layer of each cell matrix of the complex tissue organ precursor prepared by the present invention is determined by the structure and characteristics of the organ to be prepared. The synthetic polymer materials in the synthetic polymer solution used in the steps (3) and (5) are the same or different. The complex tissue organs of the present invention include artificial blood vessels, artificial hearts, artificial livers, artificial kidneys, artificial pancreas, breasts and the like.

The synthetic polymer scaffold material in the complex tissue organ precursor prepared by the invention has excellent mechanical properties, and the method of the invention can combine a plurality of different cells directly with the synthetic polymer which acts as a mechanical support to synthesize the polymer. The organic solvent in the solution is removed by extraction and does not cause significant damage to the cells. The three-dimensional structure can be directly connected to the vascular system in the body to achieve functions such as blood supply and metabolite excretion. Among them, the cell matrix solution has excellent biocompatibility, and a plurality of seed cells can form various tissues therein. The invention can realize the accurate localization of different cells/natural polymer materials and synthetic polymer scaffold materials in space, overcomes the long time required for tissue culture to induce culture cells in the three-dimensional scaffold, and the cells are unevenly distributed in the scaffold, the cells It is difficult to penetrate into the deep structure and have the disadvantages. The invention utilizes the principles of step-by-step mold/extraction method and polymer solidification forming to achieve the requirements of different cell types and structure types in different parts of complex organs, and realize reconstruction of complex tissues and organs, thereby achieving the purpose of repairing regeneration.

DRAWINGS

Figure 1 is a cross-sectional view of the mold;

1-intermediate mold, 2-internal mold

Figure 2 is a plan view of the intermediate mold and the outer mold cylinder;

Figure 3 is a cross-sectional view of the artificial heart prepared in Example 3.

3-vessel, 4-cell tissue, 5-synthetic polymer material outer layer

detailed description

The invention provides a method for preparing a complex structure organ precursor of a multilayer structure, and the specific process steps thereof are as follows: 1) dissolving the synthetic polymer material in an organic solvent to prepare a synthetic polymer solution having a mass percentage of 1% to 30%; the synthetic polymer material is polyurethane, lactic acid and glycolic acid copolymer, polylactic acid and a composite of one or two materials of a copolymer and a polyester; an organic solvent for dissolving the synthetic polymer material using tetraethylene glycol, ethylene glycol, isopropanol or 1,4-dioxane One or two anticoagulant materials of heparin, sodium citrate or paclitaxel may be added to the synthetic polymer solution, wherein the volume ratio of the anticoagulant material to the solution is 0.1 to 10%.

2) The seed cell suspension is mixed with a natural polymer solution having a mass concentration of 10% to 30% in a volume ratio of 1 to 9:9 to 1 to prepare a cell substrate solution, and the cell density of the seed cells in the solution is l xlO'-lx lO^/mL; the natural polymer is composed of one or more of gelatin, fibrinogen, collagen, chitosan, sodium alginate, hyaluronic acid and fibronectin. The mass ratio of gelatin to chitosan, gelatin and fibrinogen or gelatin and sodium alginate in the mixture of gelatin and chitosan, fibrinogen or gelatin and sodium alginate: 0.5~100: 1 The solvent for dissolving the natural polymer material is water, physiological saline, PBS solution, 0.09 M sodium chloride having a pH of 6 to 8, a solution of 3-hydroxymethylcarbamidine hydrochloride or a seed cell culture solution;

(3) injecting the synthetic polymer solution tank into a pre-designed inner mold, and then extracting the organic solvent in the synthetic polymer solution with water or PBS solution, and removing the inner mold to form a single-layer synthetic polymer material inner stent;

(4) According to the structure and characteristics of the tissue and organs to be prepared, intermediate molds of different diameters are designed, the intermediate mold is at least one, and the first intermediate mold is sleeved on the outside of the single-layer synthetic polymer material, and then the corresponding The cell matrix solution tank is injected between the inner stent and the first intermediate mold, and the natural polymer in the cell matrix solution is crosslinked by physical or chemical crosslinking, and the first intermediate mold is removed to form a stable synthetic polymer material. a two-layer structure of the intermediate layer of the stent and the first cell matrix; then placing the second intermediate mold on the outside of the two-layer structure, and then injecting the corresponding cell substrate solution into the second intermediate mold and the double Between the layer structures, the physical polymer or the chemical cross-linking method is used to crosslink the natural polymer in the cell matrix solution, and the second intermediate mold is removed to form a stable synthetic polymer material inner scaffold, the first cell matrix intermediate layer and the second cell. a three-layer structure of the intermediate layer of the matrix; the intermediate layer of the cell matrix is layer-by-layered according to the above method to form a stable multilayer structure; a glutaraldehyde solution having a concentration of 0.1 to 1% by weight, thrombin, 1 to 20 wt ° / L can be used. Calcium acid, calcium chloride or sodium triphosphate aqueous solution completes the crosslinking of natural polymers.

(5) loading the above-mentioned multilayer structure into a pre-designed outer mold, and then pouring the synthetic polymer solution into the gap between the multilayer structure and the outer mold, and extracting the outer layer of the polymer material layer with water or PBS solution. The organic solvent, which removes the outer mold, is a complex organ precursor that is made into a multilayer structure.

Preferably, the cell substrate solution is added with a cryoprotectant such as one or both of glycerin, dimethyl sulfoxide, ethylene glycol and dextran in a volume percentage of 1% to 30%. The mixture, or simultaneously or separately, is added in a volume percentage of 0.001% to 0.1% of a cell growth factor such as endothelial cell growth factor, cell transfer factor or hepatocyte growth factor.

For the preparation of adipose stem cells used in the following examples, see Cowan CM, et al. Nature Biotechnology. 2004; 22: 560-567; Yao Y, et al. Journal of Bioactive and Compatible Polymers. 2009; 24: 5-24; Other seed cells were prepared in a conventional manner. Seed cells are obtained from ex vivo tissues.

Example 1 Preparation of artificial blood vessel precursor

(1) preparing a series of cylindrical molds of different diameters, wherein the inner mold is two concentric cylinders made of stainless steel, wherein the inner cylinder is a solid body, the diameter is 2 mm, and the outer layer is a hollow cylinder. The inner diameter is 3 mm; the first and second intermediate cylindrical molds are also made of stainless steel with diameters of 4 and 5 mm respectively; the cylindrical outer mold is made of Teflon and has a diameter of 6 mm.

(2) Equipped with a concentration of 10% by weight of polylactic acid-glycolic acid copolymer (PLGA) / tetraethylene glycol

(Tetraglycol) solution, and then adding heparin to the solution, the concentration of heparin in the solution is 1% (w/v), The PLGA/tetraethylene glycol solution of the compound heparin is injected into the closed inner mold, and then the organic solvent tetraethylene glycol is extracted in distilled water to remove the inner mold to form the PLGA inner stent;

(3) preparing a fibrinogen aqueous solution having a mass concentration of 1%, and adding a human adipose tissue-derived adipose stem cell suspension to the fibrinogen aqueous solution, wherein the volume ratio of the fibrinogen aqueous solution to the adipose stem cell suspension is 9: 1 mixed, and the density of adipose stem cells in the solution is l x lO ⁵ / mL, and then hepatocyte growth factor (HGF 0.5 ng / mL), human platelet derived growth factor (BB or PDGF-BB 50) is added to the solution. Ng /mL), transforming growth factor βΐ (TGFpi lO ng / mL) and basic fibroblast growth factor (b-FGF 2.5 ng / mL), after mixing evenly, the cell matrix solution 1 is obtained; the first intermediate mold set Outside the stent in the PLGA, the cell matrix solution 1 is injected into the gap between the first intermediate mold and the PLGA stent, so that the natural polymer material is evenly distributed and the thrombin solution is injected.

(20 IU/mL) to form a stable structure of the fibrinogen/fatty stem cell layer, removing the first intermediate mold to form a double layer structure of the PLGA inner scaffold and the fibrinogen/fatty stem cell layer;

(4) preparing a gelatin/fibrinogen aqueous solution having a mass percentage concentration of 20%/2%, wherein the mass ratio of gelatin to fibrinogen is 10:1, and then adding a human blood vessel-derived endothelial cell suspension to the solution, The volume ratio of the gelatin/fibrinogen solution to the endothelial cell suspension is 1:9, and the density of the endothelial cells in the whole solution is l×10 ⁷ /mL, and the obtained mixture is the cell matrix solution 2; The second intermediate mold sleeve is external to the double layer structure obtained in the step ( ₃ ), and the cell substrate solution 2 is injected between the double layer structure and the second intermediate mold, and then the formed product is soaked with a thrombin solution (20 IU/mL) for 2 minutes to remove the first mold. The intermediate mold forms a three-layer structure of the PLGA inner scaffold, the fibrinogen/fatty stem cell thickness and the gelatin/fibrin/endothelial cell layer;

(5) The outer mold is sleeved outside the above three-layer structure, and the PLGA/Tetraglycol solution of the compound heparin prepared in the step (2) is injected between the three-layer structure and the outer mold, and the organic solvent is removed by PBS extraction.

Tetraglycol, the outer layer of PLGA is formed, and then the outer mold is removed, thereby completing the preparation of the artificial blood vessel precursor. The artificial blood vessel precursor is cultured in vitro and can be directly connected with human blood vessels to repair the defect site. Example 2 Preparation of Artificial Liver Precursor

(1) Mold material: Silicone rubber material

The structure of the inner mold: The inner mold is divided into two inner and outer layers, and the inner layer is a solid structure. The specific structure is as follows: centered on a solid cylinder with a diameter of 3 mm, and the center cylinder is the bottom side, and the center cylinder is at the two ends. Four isosceles trapezoids with a base apex at 1 cm are evenly distributed around the central cylinder. The base angle of the isosceles trapezoid is 15°, and the other three sides are solid cylinders with a diameter of 2 mm. The outer layer is a hollow structure, and The inner layer main body has the same structure, and the outer layer hollow structure can be sleeved outside the inner layer solid structure, and a gap of 1 mm is left between the two, as shown in the inner mold 2 in FIG.

The intermediate mold is placed outside the inner mold, see the intermediate mold 1 of FIG. The outer mold is placed outside the intermediate mold, and the structures of the first, second and third intermediate molds and the outer mold are the same as shown in Fig. 2, but the inner diameters are different and sequentially increased, and the inner diameters at the ports are 4.5, 5, 5.5, and 6 mm, respectively.

(2) injecting 5%% of polyurethane/ethylene glycol solution into the inner mold, and extracting by PBS to form a polyurethane inner stent;

(3) adding paclitaxel to a 2% by mass aqueous solution of fibrinogen, wherein the concentration of paclitaxel in the solution is 1% (w/v), and then adding a human blood vessel-derived endothelial cell suspension to the solution, wherein The cell density is I x ^{10 7} / mL, and the cell matrix solution 1 is obtained after uniformly mixing; the first intermediate mold is sleeved outside the polyurethane inner stent, and the cell matrix solution 1 is injected into the gap between the inner stent and the first intermediate mold. The molded article was soaked with a thrombin solution (20 IU/mL) for 2 minutes to remove the first intermediate mold to form a two-layer structure;

(4) Prepare a gelatin/fibrinogen aqueous solution having a mass percentage concentration of 10%/2%, wherein the mass ratio of gelatin to fibrinogen is 20: 1, and then the fat stem cell suspension is added to the solution, and the fat stem cells are throughout The density in the solution is I x lO ⁶ / mL, and the resulting mixture is the cell substrate solution 2; The second intermediate mold sleeve is outside the double layer structure obtained in the step (3), the cell substrate solution 2 is injected between the double layer structure and the second intermediate mold, and the formed product is soaked with a thrombin solution (20 IU/mL) for 2 minutes to remove the second mold. The intermediate mold forms a three-layer structure of a polyurethane inner stent, a fibrinogen/endothelial cell layer, and a gelatin/fibrin/fatty stem cell layer;

(5) adding the hepatocyte suspension to a 2% by mass aqueous solution of fibrinogen, wherein the density of hepatocytes is lx10 ⁷ / mL, and the obtained mixture is a cell matrix solution 3; Nested outside the three-layer structure obtained in the step (4), the cell substrate solution 3 was injected between the two, and then the molded product was soaked with a thrombin solution (10 IU/mL) for 1 minute to remove the third intermediate mold to form a four-layer structure. ;

(6) The outer mold is placed outside the above four-layer structure, and a 5%% polyurethane/ethylene glycol solution containing 5% (w/v) paclitaxel is injected between the outer mold and the four-layer structural material, and removed by PBS extraction. The mold completes the preparation of an implantable artificial liver precursor.

Example 3 Preparation of Artificial Heart Precursor

(1) Mold material: stainless steel

The structure of the inner mold: The inner mold is divided into two inner and outer layers, and the inner layer is a solid structure. The specific structure is as follows: centered on a solid cylinder with a diameter of 5 mm, and the center cylinder is the bottom side, and the center cylinder is at the two ends. Six isosceles trapezoids with a base apex at 2 cm are evenly distributed around the central cylinder. The base angle of the isosceles trapezoid is 20°, and the other three sides are solid cylinders with a diameter of 4 mm. The outer layer is a hollow structure, and The inner layer has the same main structure, and the outer layer hollow structure can be sleeved outside the inner layer solid structure, the outer center cylinder has an inner diameter of 5.5 mm, and the branch pipe is an isosceles trapezoid and the other three sides have an inner diameter of 4.5 mm.

The intermediate mold is placed outside the inner mold, see Figure 1. The outer mold is placed outside the intermediate mold, and the structures of the first, second, third and fourth intermediate molds and the outer mold are the same as shown in Fig. 2, but the inner diameters are different, and the diameters are sequentially increased, and the inner diameters at the ports are 6, 6.5, 7, and 7.5, respectively. , 8mm. (2) injecting 30%% polylactic acid/isopropanol solution into the inner mold, extracting isopropanol through PBS, removing the inner mold to form a polylactic acid inner stent;

(3) adding sodium citrate to a 1% by mass aqueous solution of collagen, wherein the concentration of sodium citrate in the solution is 1% (w/v), and then adding a human blood vessel-derived endothelial cell suspension to the solution , wherein the cell density is l xlO ⁷ cells/mL, and the cell matrix solution 1 is obtained after mixing uniformly; the first intermediate mold is sleeved outside the polylactic acid inner stent, and the cell matrix is injected into the gap between the inner stent and the first intermediate mold. Solution 1, placed at 37 ° C for 10 minutes, the collagen / endothelial cell mixture structure is stabilized, the first intermediate mold is removed to form a two-layer structure;

(4) adding a human blood vessel-derived endothelial cell suspension to a 1% collagen aqueous solution, wherein the cell density is lxlO ⁷ cells/mL, and the cell matrix solution 2 is obtained after mixing uniformly; the second intermediate mold is placed on the above double layer. Outside the structure, a cell matrix solution 2 was injected between the gaps of the two, and left at 37 ° C for 10 minutes to stabilize the structure of the collagen/endothelial cell mixture, and the second intermediate mold was removed to form a three-layer structure;

(5) adding a human-derived smooth muscle cell suspension to a 1% collagen aqueous solution, wherein the cell density is l x ^{10 7} / mL, and the cell matrix solution 3 is obtained after mixing uniformly; the third intermediate mold is placed on the above three layers. Outside the structure, a cell matrix solution 3 was injected between the gaps of the two, and left at 37 ° C for 10 minutes to stabilize the structure of the collagen/smooth muscle cell mixture, and the third intermediate mold was removed to form a four-layer structure;

(6) Adding a human-derived adipose stem cell suspension and a human cardiomyocyte suspension to a lwt% collagen aqueous solution, the ratio of the number of adipose stem cells to the neonatal rat cardiomyocytes is 1:1, and the total cell density is l xlO ⁶ / mL, after mixing uniformly, the cell substrate solution 4 is obtained; the fourth intermediate mold is placed outside the above four-layer structure, and the cell matrix solution 4 is injected between the gaps of the two, and placed in a 37 ° C incubator for 10 minutes to make the structure Stable, remove the fourth intermediate mold to form a five-layer structure;

(7) The outer mold is placed on the outside of the above five-layer structure, and a 30 wt% polylactic acid/isopropanol solution containing 3% (w/v) of sodium citrate is injected between the five-layer structure and the outer mold through PBS. Extracting organic solvent Isopropanol, the preparation of the implantable artificial heart precursor is completed after the outer mold is removed. A cross-sectional view of the artificial cardiac precursor is shown in Figure 3.

Example 4 Preparation of Kidney Precursor

(1) Mold material: PTFE

The structure of the inner mold: The inner mold is divided into two inner and outer layers, and the inner layer is a solid structure. The specific structure is as follows: centered on a solid cylinder with a diameter of 5 mm, and the center cylinder is the bottom side, and the center cylinder is at the two ends. The eight isosceles trapezoids with the base vertex at 1.5 cm are evenly distributed around the central cylinder. The base angle of the isosceles trapezoid is 30°, and the other three sides are solid cylinders with a diameter of 4 mm. The outer layer is a hollow structure. Same as the inner main body structure, the outer hollow structure can be set outside the inner solid structure, the outer center cylinder has an inner diameter of 6 mm, and the branch pipe is an isosceles trapezoid and the other three sides have an inner diameter of 5 mm.

The intermediate mold is placed outside the inner mold, see Figure 1. The outer mold is placed outside the intermediate mold. The structure of the first intermediate mold and the outer mold is the same as shown in Fig. 2, but the inner diameter is different, and the diameter is increased sequentially. The inner diameter of the port is 7,7.5 mm.

(2) injecting 30 wt% polyurethane (PU) / tetraethylene glycol solution into the inner mold, removing tetraethylene glycol by PBS extraction, and removing the inner mold to form a polyurethane inner stent;

(3) Dissolving fibrinogen and gelatin two natural biomaterials in phosphate buffer solution (PBS) to make 10wt% fibrinogen solution and 30wt% gelatin solution, then press 1:1 (v/v) The ratio is evenly mixed, then 10% dimethyl sulfoxide and 5% dextran are added to the mixture according to the volume ratio, and the human-derived adipose stem cells and the human-derived glomerular cell suspension are 1:1. The density is more uniform than that added to the above mixture to obtain a cell matrix solution 1, wherein the total cell density is lx lO ⁴ / mL; the first intermediate mold is sleeved on the outside of the polyurethane inner stent, and the inner stent and the first intermediate mold The cell substrate solution 1 was injected and fixed with a thrombin solution (30 IU/mL) for 2 minutes, and the first intermediate mold was removed to obtain a two-layer structure; (4) The outer mold is placed outside the double-layer structure, and 30% of the PU/tetraethylene glycol solution is injected between the layer structure and the outer mold, and the organic solvent is extracted by PBS, and the outer mold is removed to obtain the kidney precursor. ;

(5) Place the above kidney precursor at 4 ° C for half an hour, then -20 ° C in the refrigerator for half an hour, and finally put it into the -70 ° C low temperature refrigerator liquid nitrogen storage at low temperature, use the rapid rewarming, add commonly used The culture medium DMEM was cultured at 37 ° C under 5% CO ₂ for use.

Example 5 Preparation of Pancreatic Organ Precursor

(1) Mold material: Polyester

The structure of the inner mold: The inner mold is divided into two inner and outer layers, and the inner layer is a solid structure. The specific structure is as follows: centered on a solid cylinder with a diameter of 5 mm, and the center cylinder is the bottom side, and the center cylinder is at the two ends. Ten isosceles trapezoids with a base apex at 1 cm are evenly distributed around the central cylinder. The base angle of the isosceles trapezoid is 15°, and the other three sides are solid cylinders with a diameter of 4 mm. The outer layer is a hollow structure, and The inner layer has the same main structure, and the outer hollow structure can be sleeved outside the inner solid structure. The inner diameter of the outer center cylinder is 6 mm, and the ten branch pipes, that is, the isosceles trapezoid, the other three sides have an inner diameter of 4.5 mm.

(2) injecting 30 wt% polyester/tetraethylene glycol solution containing 3 wt% paclitaxel into the inner mold, extracting tetraethylene glycol in an organic solvent by distilled water, and removing the inner mold to form a polyester inner stent;

(3) Dissolving fibrinogen in a phosphate buffered saline (PBS) solution to prepare a 10 wt% fibrinogen solution, and then adding 20% glycerol and 5% dextran to the solution by volume ratio, by volume ratio 2: 1 The human-derived fat stem cell suspension is uniformly mixed with the human-derived islet cell suspension, and added to the 10 wt% fibrinogen solution, and the solution obtained after mixing is the cell matrix solution 1, wherein the total cell density is l xlO ⁷ / mL; the first intermediate mold is placed on the outside of the polyester inner bracket, in the polyester inner branch The cell matrix solution 1 was injected between the rack and the first intermediate mold and fixed with a thrombin solution (10 IU/mL) for 2 minutes to remove the first intermediate mold to form a double layer structure;

(4) The above double-layer structure is placed in an outer mold, and a 30 wt% polyester/tetraethylene glycol solution containing 3% of paclitaxel is injected, and the organic solvent tetraethylene glycol is extracted by distilled water, and the outer mold is removed to obtain a pancreas. Organ of organs;

(5) Place the pancreatic organ precursor at 4 ° C for half an hour, then in the refrigerator at -20 ° C for half an hour, finally put it in -196 ° C liquid nitrogen at low temperature, quickly rewarming when using, add common culture The liquid DMEM was cultured at 37 ° C under 5% CO ₂ for use.

Claims

Claim

A method for preparing a complex structure organ precursor of a multilayer structure, characterized in that the method is carried out as follows:

(2) mixing the seed cell suspension with a natural polymer solution having a mass concentration of 10% to 30% to prepare a cell matrix solution, wherein the density of the seed cells in the cell matrix solution is l xlO^l xlO ⁹ /mL _;

(4) According to the structure and characteristics of the tissue and organs to be prepared, intermediate molds of different diameters are designed, the intermediate mold is at least one, and the first intermediate mold is sleeved on the outside of the single-layer synthetic polymer material, and then the corresponding The cell matrix solution tank is injected between the inner stent and the first intermediate mold, and the natural polymer in the cell matrix solution is crosslinked by physical or chemical crosslinking, and the first intermediate mold is removed to form a stable synthetic polymer material. a two-layer structure of the intermediate layer of the stent and the first cell matrix; then the second intermediate mold is sleeved on the outside of the two-layer structure, and the corresponding cell matrix solution tank is injected between the second intermediate mold and the two-layer structure, via physical Or chemical crosslinking method, cross-linking the natural polymer in the cell matrix solution, removing the second intermediate mold to form a stable synthetic polymer material inner scaffold, the first cell matrix intermediate layer and the second cell matrix intermediate layer three-layer structure Increasing the intermediate layer of the cell matrix layer by layer according to the above method to form a stable multilayer structure;

(5) loading the above-mentioned multilayer structure into a pre-designed outer mold, and then pouring the synthetic polymer solution into the gap between the multilayer structure and the outer mold to form an outer layer of synthetic polymer material; (6) extracting the organic solvent in the outer layer of the synthetic polymer material with water or PBS solution, and removing the outer mold, thereby preparing a complex structure organ precursor of a multilayer structure.

The method for preparing a complex structure organ precursor of a multilayer structure according to claim 1, wherein the synthetic polymer material is polyurethane, a copolymer of lactic acid and glycolic acid, polylactic acid and a copolymer thereof, and A composite of one or both of the polyesters.

The method for preparing a complex structure organ precursor of a multilayer structure according to claim 1, wherein: the natural polymer is gelatin, fibrinogen, collagen, chitosan, sodium alginate, and transparent At least one of a phytic acid and a fibronectin.

The method for preparing a complex structure organ precursor of a multilayer structure according to claim 1, wherein the organic solvent for dissolving the synthetic polymer material in the step (1) is tetraethylene glycol or B. Glycol, isopropanol or 1,4-dioxane.

The method according to claim 1, wherein the solvent of the natural polymer solution in the step (2) is water, physiological saline, PBS solution, pH = 6 to 8 of 0.09 M sodium chloride, 3-hydroxymethylaminomethane hydrochloride solution or cell culture solution.

The method for preparing a complex structure organ precursor of a multilayer structure according to claim 1, wherein: the cell substrate solution further comprises a cryopreservation agent in a volume percentage of 1% to 30%.

The method for preparing a complex structure organ precursor of a multilayer structure according to claim 6, wherein: the cryoprotectant is one of glycerin, dimethyl sulfoxide, ethylene glycol and dextran. Kind or a mixture of two materials.

The method for producing a complex structure organ precursor of a multilayer structure according to claim 1 or 6, wherein the cell substrate solution is added in a volume percentage of 0.001% to 0.1% of cell growth factor.

The method for preparing a multi-layered complex tissue organ precursor according to claim 8, wherein: the cell growth factor is endothelial cell growth factor, cell transfer factor or hepatocyte growth factor Child.

The method for preparing a complex structure organ precursor of a multilayer structure according to claim 1 or 6, wherein: one or two anticoagulations are further added to the synthetic polymer solution or the cell matrix solution. The material, wherein the volume ratio of the weight of the anticoagulant material to the solution is 0.1 to 10%.